Immunoglobulin igg3 as a marker for protecting against infectious viral diseases, and the uses of the same

ABSTRACT

The invention relates to a novel variant of isolated and/or purified immunoglobulin IgG3 which can be used as a marker for protecting against infectious viral diseases such as AIDS, as a diagnostic tool, or as a preventive and curative medicament. The invention also relates to corresponding in vitro diagnostic methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national phase entry under 35 U.S.C. § 371 ofInternational Application No. PCT/FR03/00463 filed Feb. 13, 2003, whichclaims priority from French application 0201960, filed Feb. 25, 2002.

FIELD OF THE INVENTION

The present invention relates to the field of biology and moreparticularly concerns a new variant of isolated and/or purifiedimmunoglobulin IgG3, useful as a protective marker for infectious viraldiseases such as AIDS, and as a diagnostic tool or as a preventative andcurative medicament.

BACKGROUND OF THE INVENTION

AIDS is an extremely serious and worrying public health problem for manycountries throughout the world. In the USA, for example, the officialnumber of AIDS cases is greater than 100,000 and the number of peopleinfected has been estimated at more than 1 million. The propagation ofthe disease is accentuated by the number of chronic bearers of the virusresponsible for AIDS who remain asymptomatic for many years, or eventheir entire life, and are therefore unidentified sources of infection.This disease, which can be transmitted by sexual contact and via theblood, affects the immune system of the host, thus causing theappearance of opportunistic infections or of pathologies against whichthe host would have been protected by a healthy immune system. Once AIDShas been recognized, death usually occurs two to three years afterdiagnosis following a breakdown of the patient's immune defences andmultiple opportunistic infections. It is very difficult to classify theAIDS viruses given the extreme genetic and antigenic variability theyexhibit; conventionally, it is acknowledged that there are two types ofvirus responsible for human AIDS: HIV-1 and HIV-2 (humanimmunodeficiency virus). For a number of reasons, the HIV replicationmechanism poses many problems in terms of obtaining effective treatment.This is because proviral DNA, on account of its integration in the cellgenome, behaves likes a genetic element of the host. Moreover, the HIVvirus is disseminated throughout the entire body in the T lymphocytes,the monocytes, the macrophages and in the central nervous system.Finally, the HIV virus has extremely high antigenic variability. Thevarious curative therapies currently used in clinical medicine consistessentially either in blocking the activity of reverse transcriptase orin inhibiting the activity of viral enzymes that are indispensable forinfection or replication (proteases, integrases). The effectiveness ofthese therapies remains limited since the absorption of these antiviralscauses side effects. Moreover, given the high rate of mutation of theHIV virus, the latter rapidly becomes resistant to drugs, such as AZTand other nucleotide analogues, during therapy. The emergence ofresistant strains makes it necessary to increase the therapeutic dosesadministered to patients. Failure of current curative therapiestherefore means that it is necessary to develop new therapeuticstrategies for fighting retroviral infection.

In their research to develop new curative strategies, the inventors havebeen led to study the HIV-positive populations who are infected with HIVbut are non-progressors, that is to say have not developed AIDS. Theyhave thus been able to show for the first time that in theseHIV-positive patients who are infected with HIV classified asnon-progressors there is a particular variant of IgG3. The inventorshave shown that this variant of IgG3 differs by its primary structure oflower molecular weight, its longer half-life and its higher serumconcentration than in the case of conventional IgG3s. The immunoglobulinIgG3 demonstrated by the inventors, which has been isolated andpurified, appears to be a protective marker for AIDS and an agent whichneutralizes the causative agent of this disease.

Human immunoglobulin IgGs can be divided into four subclasses (IgG1,IgG2, IgG3, IgG4) which differ by minor differences in the primarystructure of their heavy chain. The main differences concern the hingeregion and the number of inter-chain disulphide bridges. Thus, the hingeregion of IgG3 is very long, which accounts for its higher molecularweight (170 kDa) and certain biological properties such as its half-life(in days) which is much shorter for immunoglobulin IgG3s (7 days) thanfor the other IgG subclasses which have a half-life of around 20 days.IgG3s are capable of selectively binding to certain receptors to the Fcfragment of immunoglobulins such as RFcγI, RFcγIIa, RFcγIIIa.Immunoglobulins, the primary function of which is to bind the antigen soas to neutralize it, also have the role of activating secondary effectorfunctions in particular by way of the complement. The complement system,which is a complex group of serum proteins involved in inflammatoryreactions, is one of the most important effector mechanisms for humanIgG1s, IgG3s and IgGMs. After having bound the antigen, the IgG1s, IgG3sand IgGMs can activate the enzymatic cascade in the conventional mannerof the complement known as C1q, IgG2s for their part being relativelyineffective and IgG4s being incapable of doing so. The binding of C1q tothe immunoglobulin is the first step in the cascade of the complementwhich leads to cell lysis. This mechanism is particularly important forfighting infectious and viral diseases such as AIDS, for example, andplays a crucial role in destroying infected cells.

SUMMARY OF THE INVENTION

The work carried out by the inventors thus makes it possible to meet anurgent need, namely the need to develop new preventive and/or curativemedicaments or compositions intended to neutralize the AIDS virus, andalso to develop new diagnostic tests which make it possible to adapt thetherapy as a function of the serotype of the patient.

The subject of the present invention is therefore a purified and/orisolated human immunoglobulin of class IgG3, or one of the fragmentsthereof, having the following characteristics (i) a life span in theserum of the patient which is greater than the life span of the IgG3snormally present in human serum and in particular of at least 15 days,preferably of at least one month, (ii) a heavy chain, the molecularweight of which, determined by electrophoretic mobility, is less thanthe molecular weight of the IgG3s normally present in human serum (60kDa), said heavy chain having a molecular weight of about 50 kDa andcomprising the complement binding site. This reduction in the apparentmolecular weight determined by electrophoresis reflects either areduction in length of the primary structure of the IgG3 or a differentthree-dimensional shape or a reduction in the number ofpost-translational modifications such as, for example, the glycosylationof the heavy chain.

The variant immunoglobulin IgG3 according to the invention in serum isfinally characterized by a serum concentration that is at least once asgreat, at least twice as great, in general at least three times as greatas the IgG3 concentration of a normal serum, which represents a serumconcentration of this new IgG3 variant of about 1 g/L.

According to one embodiment of the invention, the variant immunoglobulinIgG3 is isolated and/or purified from HIV-positive patients; thesepatients are non-progressors, that is to say people who have beeninfected with HIV for a number of years without any loss of immunedefence, and who are thus asymptomatic. More preferably, theimmunoglobulin IgG3 according to the invention selectively binds the R7Vepitope of the gp160 protein of the HIV-1 virus, said epitope comprisingthe sequence Arg-Thr-Pro-Lys-Ile-Gln-Val.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1E are photographs of electrophoretic gels; FIG. 1A shows acomparison of an IgG₃ of a normal serum (SN1) with that of anon-progressor HIV-positive patient (ARG); FIG. 1B shows a confirmationof the lower molecular weight of the heavy chain with another patient(ETC); FIG. 1C shows other patients (THO, GEM); FIG. 1D shows otherpatient (VAL) and other HIV-negative person (SN2); and FIG. 1E showsother patient (BOI)

FIG. 2 is a diagram that shows a protocol for immunoprecipitation of theHIV retrovirus with the variant IgG3 by way of example.

FIG. 3 is a bar graph that shows immunoprecipitation of the virus withthe IgG₃s of three different patients.

FIG. 4. is a bar graph that shows immunoprecipitation of variableamounts of virus with a fixed amount of IgG3 antibody.

FIG. 5. is a bar graph that shows immunoprecipitation of differentstrains with a fixed amount of IgG3.

FIG. 6 is a diagram summarizing the virus neutralization protocol.

DETAILED DESCRIPTION

In the present description, the terms immunoglobulin and antibody willbe used without distinction. The term epitope is intended to mean anydeterminant of the protein responsible for specific interaction with theantibody; epitopes usually consist of groups of molecules havingchemically active surfaces such as amino acids or side chains of sugarsand having a specific three-dimensional structure and/or a specificcharacteristic charge.

The methods of isolating and/or purifying immunoglobulins, the methodsfor determining the length of the heavy chain of immunoglobulins and themethods of determining the life span of IgG3s in serum are known to theperson skilled in the art working in the field of immunology. By way ofillustration, various methods and protocols which can be used aredescribed in “Current Protocols in Immunology”, updated annually (4volumes), published by the “National Institutes of Health”, by John E.Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, WarrenStrober—Wiley Interscience.

By way of illustration, the purification of the IgG3s of the presentinvention is carried out using immunoaffinity columns Hitrap™ Protein GHP (ref. 17-0404-01) and Hitrap™ Protein A HP (ref. 17-0402-01) fromAmersham-Pharmacia. Once the serum/plasma has passed through the Hitrap™Protein A HP column, which binds only the IgG1s, IgG2s and IgG4s, theserum/plasma which has had the IgG1s, IgG2s and IgG4s removed is passedthrough the Hitrap™ Protein G HP column in order to purify the IgG3s.

The molecular weight of the heavy and light chains of the IgGs isdetermined for example by electrophoresis on a 12% polyacrylamide gel(Ready Gel Tris-HCl glycine BIORAD ref. 161-0901). In brief, the IgG3spurified on the immunoaffinity column are reduced by heating for 3minutes at 95° C. in the presence of a reducing buffer (Laemmli sampleBuffer BIORAD ref. 161-0737 +β mercaptoethanol) then loaded onto thegel. Migration takes place within 40 minutes at 200 V, 35 mA. The gelsare dyed using a commercial solution (Bio-Safe Coomassie BIORAD, ref.161-0786).

Finally, the life span of the variant IgG3s is determined by an ELISAtest carried out each month or each quarter in the serum or plasma ofnon-progressor patients, making it possible to confirm the presence ofIgG3.

By isolation of the variant immunoglobulin IgG3 according to theinvention, the preparation of monoclonal antibodies of variant IgG3 typeis also covered. In order to prepare monoclonal antibodies or fragmentsthereof, reference may be made to the techniques which are described inparticular in the manual “Antibodies” (Harlow et al., 1988) or to thetechnique for preparation from hybridomas described by Kohler andMilstein in 1975. The monoclonal antibodies according to the inventionmay be obtained for example from cells of an animal immunized againstthe gp160 protein, or one of the fragments thereof, and comprising theR7V epitope. The antibody according to the invention may be purified forexample on an affinity column on which the target epitope or antigen hasbeen immobilized beforehand, for example the R7V epitope of the gp160protein of HIV.

The inventive concept on which the invention is based cannot be reducedsolely to the immunoglobulin IgG3s purified and/or isolated frompatients suffering from AIDS. This is because the variant immunoglobulinIgG3s demonstrated by the inventors are also expressed in the serum ofpatients suffering from other pathologies involving a viral infection.Among these pathologies involving a viral infection, mention may be madeby way of non-exhaustive examples of the pathologies associated withhuman T-cell leukaemia viruses (HTLV), the cytomegalovirus (CMV), herpesviruses (HSV-1, HSV-2), the Epstein Barr virus (EBV) and hepatitisviruses (HBV, HBC). The invention also relates to the variantimmunoglobulin IgG3s isolated and/or purified from a patient sufferingfrom an infectious disease of variable development, for instanceneurological diseases such as multiple sclerosis, skin diseases such aspsoriasis, auto-immune diseases such as systemic lupus erythematosus,and cancers of retroviral origin such as familial inflammatory breastcancer.

The invention also covers the fragments of variant immunoglobulin IgG3.The antibody fragments according to the invention comprise any fragmentof said antibody capable of binding to the epitope of the protein towhich there binds the antibody from which said fragment comes. Examplesof such fragments include in particular single-chain antibodies ormonovalent fragments Fab or Fab′ and divalent fragments such as F(ab′)2,which have the same binding specificity as the antibody from which theycome. A fragment according to the invention may also be a single-chainFv fragment. Antibody fragments of the invention may be obtained fromimmunoglobulins according to the invention by methods such as digestionby enzymes, such as pepsin or papain, and/or by cleavage of thedisulphide bridges by chemical reduction. Alternatively, followingsequencing of the primary structure of the variant IgG3 according to theinvention, the antibody fragments may be synthesized by automaticpeptide synthesizers such as those supplied by the company AppliedBiosystems for example.

The present invention also relates to antisera comprising theimmunoglobulin according to the invention.

The person skilled in the art has at his disposition the molecular andcell biology tools for carrying out the cloning, sequencing andexpression, by the recombinant route, of the variant immunoglobulinIgG3s (Sambrook et al., 1989; Coligan et al., Current Protocols inImmunology, see page 6). These recombinant IgG3s, which are also withinthe scope of the present invention, could thus be produced in vitro andoptionally modified by recombinant DNA technologies or chemistrytechnologies in order to give them particular properties.

Depending on the desired diagnostic applications, the antibody accordingto the invention or one of the fragments thereof may be immobilized on asupport. The immobilization or coupling may be carried out on manysupports known to the person skilled in the art. This immobilization orcoupling is preferably carried out on a solid support either directly orindirectly via a spacer arm. The solid supports may in particularinclude glass, polystyrene, polypropylene, polyethylene, dextran, nylon,or natural or modified celluloses. These supports may be either solubleor insoluble.

For other applications, the antibody according to the invention or oneof the fragments thereof may be labelled directly or indirectly by amarker so as to obtain a conjugate that makes it possible to generate adetectable and/or quantifiable signal that can be used for in vivo or invitro diagnosis. The corresponding diagnostic kit comprising thelabelled antibody, or one of the fragments thereof, is also one of thesubjects of the present invention. The marker may be selected fromenzymes, dyes, haptenes, luminescent agents such as radioluminescent,chemiluminescent, bioluminescent, fluorescent and phosphorescent agents,ligands such as biotin, avidin, streptavidin, digoxigenin, andradioactive isotopes. Thus, the immunoglobulin according to theinvention or one of the labelled fragments thereof is conjugated forexample with enzymes such as peroxidase, alkaline phosphatase,β-D-galactosidase, glucose oxidase, glucose amylase, carbonic anhydrase,acetyl cholinesterase, lysozyme, malate dehydrogenase orglucose-6-phosphate dehydrogenase, or with a molecule such as biotin,digoxigenin or 5-bromo-deoxyuridine. Fluorescent markers may also beconjugated to the immunoglobulin or one of the fragments thereofaccording to the invention and include for example fluorescein and itsderivatives, rhodamine, Texas red, dansyl, umbelliferone andautofluorescent proteins such as GFP (“Green Fluorescent Protein”), etc.Other conjugates may also include chemiluminescent markers such asluminol and dioxetanes or bioluminescent markers such as luciferase andluciferin.

Among the radioactive isotopes which can be bound to the immunoglobulinor one of the fragments thereof according to the invention, preferenceis also given to the radioactive markers such as ¹⁴C, ³⁶Cl, ⁵⁷Co, ⁵⁸Co,⁵¹Cr, ¹⁵²Eu, ⁵⁹Fe, ³H, ¹²⁵I, ¹³¹I, ³²P, ³⁵S, ⁷⁵Se and ^(99m)Tc, whichcan be detected by known means such as the gamma-ray counter orscintillation counter, by autoradiography, etc. The present inventionalso comprises the conjugates whose detectable marker is selected fromthe markers which can be used in in vivo imaging. Examples of suchmarkers according to the invention are ⁷²As, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ¹²³I,¹²⁵I, ¹³¹I, ¹¹¹In, ⁹⁷Ru, ^(99m)Tc, ²⁰¹Tl and ⁸⁹Zr. The invention alsoincludes the paramagnetic isotopes used in magnetic resonance imaging(MRI), which include in particular ⁵²Cr, ¹⁶²Dy, ⁵⁶Fe, ¹⁵⁷Gd and ⁵⁵Mn.The term “in vivo imaging” must be understood in the present descriptionto mean any method which makes it possible to detect a labelled antibodyaccording to the present invention or one of the fragments thereof whichbinds specifically to the epitope of the protein of interest in the bodyof the patient. The patient will preferably be a man liable to havecells infected by a virus or suffering from an infectious disease ofvariable development, abnormally expressing the protein of interest.

Such conjugates may be prepared by methods known to the person skilledin the art. They may be coupled to the markers directly or by way of anintermediate functional group, a spacer group or a linkage group such asa polyaldehyde, for instance glutaraldehyde, ethylenediamine-tetraaceticacid (EDTA), diethylenetriaminepentaacetic acid (DPTA), or in thepresence of coupling agents such as periodate, benzoquinone, etc.Conjugates comprising markers of fluorescein type may be prepared byreaction with an isothiocyanate.

One of the objects of the present invention is also to provide a methodfor the diagnosis and/or in vitro dosage of a pathology selected fromviral infections and infectious diseases of variable development,comprising the steps (i) contacting a sample of body fluid and/or bodytissue, from a patient in which the presence of said pathology issuspected, with an immunoglobulin, a fragment of immunoglobulin or anantiserum according to the invention; and (ii) detecting the presence ofan immune complex between said immunoglobulin and a specific epitope ofsaid pathology. The term body fluid or biological fluid is understood tomean fluids such as serum, total blood, urine, sperm, cells, a tissuesample or biopsies of human origin. There are many methods for detectingthe immune complex, these being known to the person skilled in the art;they depend on the nature of the diagnosis and the dosage to be carriedout. This may be the ELISA test, RIA, a sandwich method, animmunoprecipitation, an agglutination test, a competition test, or anytest known to the person skilled in the art which depends on theformation of an antibody-antigen immune complex. By way of example, onepreferred method uses immunoenzymatic processes according to the ELISAtechnique, immunofluorescence processes, radioimmunological (RIA)processes, or equivalents thereof. All these methods are preferablybased on the binding of the immunoglobulins in question to antigenpeptides and then the demonstration of this binding.

More particularly, the invention also covers a method for the diagnosisand/or in vitro dosage of the human acquired immune deficiency syndrome(AIDS), comprising the steps (i) contacting a sample of body fluidand/or body tissue, from a patient in which the presence of HIV issuspected, with an immunoglobulin, a fragment of immunoglobulin or anantiserum according to the invention, and (ii) detecting the presence ofan immune complex between said immunoglobulin and the gp160 protein ofHIV, and in particular the R7V epitope of this protein contained in saidsample.

The invention also relates to an in vitro or in vivo method for treatingbiological fluid of a patient suffering from a pathology selected fromviral infections, in particular HIV, and infectious diseases of variabledevelopment, said method comprising the step of contacting an effectiveamount of immunoglobulin, a fragment of immunoglobulin or an antiserumaccording to the invention with said biological fluid, so as toneutralize a specific epitope of the pathology in said fluid. The methodmoreover optionally comprises the additional step of removing from thereaction mixture the immune complex thus formed. Within the context ofan in vitro method for treating biological fluid, the method mayfurthermore comprise the additional step of reinjecting into the patientall or part of the biological fluid thus treated. Neutralization of theHIV virus, for example, is understood to refer to any mechanism whichhas the in vivo effect of destroying and/or preventing the propagationof viruses. The variant immunoglobulin IgG3 according to the inventionappears to constitute, given its G3 subtype and its stability, anexcellent means for neutralizing any body fluid intended to bereinoculated or reintroduced into an individual, such as the blood of anHIV-positive man for giving a blood transfusion to an individual, atissue of an HIV-positive man in the case of a tissue graft, or thesperm of an HIV-positive man for inseminating an HIV-negative woman.This is because, since the IgG3 subtype binds the complement, theimmunoglobulins according to the invention induce the lysis of theinfected cells.

According to one particular embodiment, the invention provides an invitro method for neutralizing cells infected with HIV in a biologicalsample of body fluid and/or body tissue from an HIV-positive patient.This method comprises the following steps:

-   -   (i) combining simultaneously, separately or sequentially the        immunoglobulin or one of the fragments thereof or the antiserum        according to the invention with the biological sample containing        the cells infected with HIV having the gp160 protein at their        surface and with protein G bound to magnetic particles;    -   (ii) incubating the mixture obtained in (i) under conditions        which allow the binding of said immunoglobulin to the gp160        protein, and preferably to the R7V epitope, so as to form a        complex, said complex comprising said antibody bound to an        HIV-infected cell fixed to this magnetic particle;    -   (iii) displacing this magnetic particle to a predetermined point        of the container containing this reaction mixture, such that the        displacement is brought about by a magnetic field acting on said        magnetic particle.

The invention also covers an in vitro method for diagnosing anon-progressor patient suffering from a pathology selected from viralinfections, in particular AIDS, and infectious diseases of variabledevelopment, characterized in that the presence of immunoglobulin IgG3is detected, preferably by means of an immunological test, saidimmunoglobulin IgG3 having the following characteristics (i) a life spanin the serum of the patient that is greater than that of the IgG3s thatare normally present, and in particular of at least 15 days, preferablyat least one month, (ii) a heavy chain, the molecular weight of which,determined by electrophoretic mobility, is less than the molecularweight of the IgG3s normally present in human serum (60 kDa), said heavychain having a molecular weight of about 50 kDa and comprising thecomplement binding site, and (iii) optionally having a serumconcentration that is at least once as great, at least twice as great orat least three times as great (i.e. 1 g/L) as the IgG3 concentration ofa normal serum. The diagnostic test used is preferably selected from anELISA test, RIA, a sandwich method, an immunoprecipitation or anagglutination test. This diagnostic method for detecting the IgG3variant according to the invention is of considerable interest. This isbecause the HIV-positive patients who carry this IgG3 variant arenon-progressors. In this case, diagnosis is very favourable and it ispossible to avoid heavy therapeutic treatments. This is particularlytrue in the case of a pregnancy where the presence of these antibodiesin the mother (HIV+) appears to lead to non-infection in then newbornchild. The present invention therefore aims to use the variantimmunoglobulin IgG3 or one of the fragments thereof or the antiserumaccording to the invention as a protective marker in pathologiesselected from viral diseases, in particular AIDS, and infectiousdiseases of variable development.

More generally, the present invention aims to cover the use of animmunoglobulin or one of the fragments thereof or an antiserum accordingto the invention to carry out an immunological test selected from theELISA test, the RIA test, the sandwich method, immunoprecipitation orthe agglutination test.

The present invention also aims to cover the use of the immunoglobulinaccording to the invention as a medicament, and more precisely as atherapeutic antibody or as a targeting agent. The term therapeuticantibody is understood to mean immunoglobulin IgG3s or a serum or plasmacontaining them, injected intravenously into progressor patients inrespect of whom conventional therapy is generally not successful.

The variant immunoglobulin IgG3 or the antiserum containing it can beused to prepare a medicament intended for the therapeutic treatmentand/or prophylaxis of a viral disease, in particular AIDS, but alsodiseases of which the causative agent is a virus as listed above. Whenthe disease is AIDS, the variant immunoglobulin IgG3 used to prepare amedicament intended to neutralize the HIV gp160 protein in a mansuffering from AIDS is preferably directed against the R7V epitope ofthe gp160 protein of HIV. The variant immunoglobulin IgG3 or theantiserum containing it can also be used to prepare a medicamentintended for the therapeutic treatment and/or prophylaxis of aninfectious disease of variable development.

The immunoglobulin IgG3 or one of the fragments thereof according to theinvention may be used as a medicament in the form of a mixture with atleast one anti-retroviral agent selected from the group comprisingreverse transcriptase and/or viral antiprotease inhibitors as acombination product for simultaneous use, separate use or sequentiallyuse in antiviral therapy. The reverse transcriptase inhibitor ispreferably selected from 3′-azido-3′-deoxythymidine (AZT),2′,3′-dideoxyinosine (ddI), 2′,3′-dideoxycytidine (ddC),(−)2′,3′-dideoxy-3′-thiacytidine (3TC),2′,3′-didehydro-2′,3′-dideoxythymidine (d4T) and(−)2′-deoxy-5-fluoro-3′-thiacytidine (FTC), TIBO, HEBT, TSAO, α-APA,nevirapine, BAHP, phosphonoformic acid (PFA); the viral antiprotease isselected from indinavir and saquinavir.

In the context of a use of the antibody as a targeting agent, saidantibody is optionally modified by chemistry technologies and/orrecombinant DNA technologies, in order to modify its stability, itsaffinity, its bioavailability or its compatibility, etc. Theimmunoglobulin according to the invention as a targeting agent isconjugated directly or indirectly to at least one agent selected fromthe group comprising antiproliferative agents, antineoplastic agents orcytotoxic agents. Included among the agents which may be conjugated tothe antibodies according to the invention are, besides the radioisotopesand the markers described above in particular, alkylating compounds suchas mechlorethamine, triethylene phosphoramide, triaziquone, camustine,semustine, methotrexate, mercaptopurine, cytarabine, fluorouracil,antibiotics such as actinomycin, hormones or hormone antagonists such ascorticosteroids, for instance prednisone or progestins, bacterial orviral toxins. In one particular embodiment, the non-isotopicantiproliferative and/or antineoplastic and/or cytotoxic agent is anucleic acid molecule such as, for example, single-stranded DNA,double-stranded DNA, single-stranded RNA, in particular an antisenseRNA, double-stranded RNA or an RNA/DNA hybrid. This nucleic acidoptionally encodes for a protein product of interest.

It also falls within the scope of the invention to provide a vaccine orpharmaceutical composition for the treatment or prevention of apathology selected from viral infections and infectious diseases ofvariable development, comprising an immunoglobulin antibody or anantiserum according to the invention in association with apharmaceutically acceptable carrier, excipient or diluent. Thepharmaceutically acceptable excipients include, in particular, water,saline solutions, buffers or any other compound described for example inthe Merck Index.

The compositions or vaccine according to the invention may also comprisecomponents which increase the immunogenicity, in particular specific ornon-specific immunity adjuvants such as Freund's adjuvant,polysaccharides or equivalent compounds. These are compounds known tothe person skilled in the art in the field of vaccination. Thecompositions may be in any form whatsoever that is compatible with theselected route of administration. The administration of these compoundsto the patient may be local or systemic and may be carried out by theintravenous, intra-arterial, intramuscular or intraperitoneal route, byway of the spinal fluid, or by the intradermal, oral, nasal, anal route.However, the compositions according to the present invention could beused by other routes, in particular by the aerosol route, in order toinduce protection of the mucous membranes.

The amount of variant immunoglobulin IgG3s, or one of the fragmentsthereof, comprised in the pharmaceutical compositions according to thepresent invention and necessary for effective therapy will depend onvarious factors such as the mode of administration, the part of the bodytargeted, the physiological condition and the age of the patient, anyside effects, counter-indications if there are any, concomitanttherapies or other variables that a person skilled in the art will knowhow to adjust. The dosage for such therapeutic prevention or treatmentwill be carried out in such a way as to optimize the safety andeffectiveness thereof. In general, the dosages used in vitro may providean indication of the quantities used for administering the antibody insitu and thus use may be made of animal models to determine theeffective quantities of immunoglobulins according to the invention forthe treatment of a particular pathology, in particular AIDS.

The monoclonal antibodies or fragments thereof according to theinvention also constitute a means of immunocytochemical orimmunohistochemical analysis for analysing the expression of the proteinof interest on electrophoresis gels or transfer membranes or specifictissue sections, for example by immunofluorescence or by radioactive orgold enzyme labelling. They make it possible in particular todemonstrate and quantify the specific normal or abnormal presence of theprotein of interest in tissues or biological samples, which makes themuseful for identifying and locating the expression of the protein ofinterest, for diagnosing pathologies associated with the abnormalpresence of the protein of interest, such as for example gp160, but alsofor monitoring the development of methods for preventing or treating apathology requiring said detection or said dosage. More generally, theantibodies, the fragments thereof or the antiserum according to theinvention may advantageously be used in any situation where theexpression of a protein of interest, for example the gp160 proteincontaining the R7V epitope, must be observed in a qualitative and/orquantitative manner.

Other features and advantages of the invention will emerge from thefollowing description with the examples and the figures, the legends ofwhich are given below.

EXAMPLES Example 1 Demonstration of IgG3 Variants in Certain IndividualsInfected with HIV and Qualified as Non-Progressors

The immunoglobulin antibodies are divided into four subclasses, andgenerally the antibodies induced after immunization belong to the IgG₁s,which have a very long half-life.

The inventors describe here, in patients infected with HIV, the presenceof protective antibodies associated with IgG₃s, whereas the antibodieswhich indicate infection are always IgG₁s. An IgG₃ variant exists insome individuals who are infected with HIV and are qualified asnon-progressors.

1.1. The IgG3 Variant has a Life Span of Several Months

This variant differs from the conventional IgG₃s in terms of itshalf-life; the half-life is seven days for conventional IgG₃s andseveral months for the variant, as shown by the study on the patientsequential follow-ups, the serums being sampled at intervals of severalmonths (Table 1).

Table 1 shows the results of an ELISA test searching for protectiveantibodies directed against an epitope associated with HIV, called R7V.These antibodies are revealed by second antibodies which distinguish thevarious subclasses of human immunoglobulins.

The anti-R7V protective antibodies are always IgG₃s and/or IgG₄s, butnever IgG₁s or IgG₂s, and their high titre remains over several months.TABLE 1 Study of the anti-R7V IgG subclasses of sequential serums Datessamples were taken IgG₁₊₂ IgG₃ IgG₄ Positivity threshold = 0.1 Serum ofpatient 1 diluted ⅕ L Blank 0.075 0.117 0.127 Seronegative −0.005 0.031−0.012 control 02/12/1993 −0.004 0.002 −0.011 05/04/1994 −0.002 0.2160.025 28/07/1994 0 0.456 0.028 10/11/1994 −0.004 0.070 0.029 09/03/19950.0015 0.262 0.042 13/07/1995 −0.004 0.130 0.035 22/02/1996 −0.009 0.2240.032 20/06/1996 −0.007 0.128 0.025 28/11/1996 −0.006 0.166 0.05003/03/1997 −0.003 0.110 −0.065 05/06/1997 −0.005 0.085 −0.069 15/12/1997−0.007 0.073 −0.069 11/05/1998 −0.008 0.090 −0.069 25/09/1998 −0.0020.153 −0.063 11/10/1999 −0.004 0.148 −0.067 11/01/2001 −0.006 0.133−0.069 Positivity threshold = 0.1 Serum of patient 2 diluted ⅕ L Blank0.075 0.117 0.127 Seronegative −0.005 0.031 −0.012 control 01/07/1993−0.004 0.565 −0.068 11/10/1993 −0.003 0.381 −0.066 17/01/1994 −0.0020.418 −0.065 02/05/1994 −0.006 0.442 −0.068 01/08/1994 −0.008 0.419−0.069 05/12/1994 −0.003 0.455 −0.062 10/04/1995 −0.005 0.440 −0.06503/07/1995 −0.007 0.397 −0.068 30/10/1995 −0.004 0.726 −0.072 29/01/19960 0.641 −0.065 06/05/1996 −0.006 0.544 −0.068 19/09/1996 −0.007 0.491−0.067 20/01/1997 −0.009 0.374 −0.064 06/10/1997 −0.002 0.394 −0.06412/01/1998 −0.005 0.558 −0.066 19/03/1998 −0.007 0.532 −0.068 22/06/1998−0.007 0.503 −0.074 28/09/1998 −0.004 0.457 −0.062 01/02/1999 −0.0060.230 −0.067 26/04/1999 −0.01 0.086 −0.064 15/07/1999 −0.013 0.060−0.066 16/12/1999 0.099 0.079 −0.061 23/03/2000 0.084 0.058 −0.064

1.2. The IgG3 Variant has a Heavy Chain of Approximately 50 kDa

The electrophoretic analyses of FIG. 1 show that the IgG₃ variantpresent in non-progressor HIV-infected patients has a heavy chain withgreater electrophoretic mobility, which means that its molecular weightis lower than that of a conventional IgG₃ purified in a normalHIV-negative subject.

FIGS. 1 a to 1 e show the migration of the IgG₃s purified either fromnormal HIV-negative subjects or from HIV-positive patients on a 12%polyacrylamide gel. The heavy chains have a molecular weight of around50 kDa, whereas the light chains have a molecular weight of 25 kDa.(Molecular weight markers: M₁: 250; 150; 100; 75; 50; 37; 25; 15; 10kDa/M₂: 200; 116; 97; 66; 45; 31; 21.5; 14.4; 6.5 kDa).

The IgG₃ variant is recognized by antibodies directed either against theFab part of the IgG₃s or antibodies directed against the hinge part. Thevariation should therefore concern the Fc fragment, probably at theglycosylation level.

1.3. The IgG₃ Variant is Found in Most Non-Progressor HIV-PositivePatients (Table 2). TABLE 2 Determination of the anti-R7V IgG subclassby ELISA Positivity threshold = 0.2 IgG₁ IgG₂ IgG₃ IgG₄ Blank 0.0580.056 0.085 0.081 Seronegative control 1/50 0.059 0.060 0.113 0.091Positive control 0.057 0.057 0.854 0.084 1 ALB HE 1/50 (07/01/94) 0.0720.063 0.080 0.073 2 ANO MA 1/50 (13/11/00) 0.061 0.057 0.136 0.108 3 ARGCH 1/50 (30/11/00) 0.058 0.055 0.481 0.071 4 AUC LA 1/50 (15/11/99)0.065 0.061 0.112 0.138 5 AUR PH 1/50 (03/01/94) 0.069 0.064 0.343 0.6856 BEN MI 1/50 (14/02/00) 0.062 0.064 0.077 0.071 7 BER YV 1/50(13/03/00) 0.105 0.073 0.109 0.307 BER YV 1/50 (25/11/99) 0.102 0.0720.107 0.339 8 BITT MA 1/50 (27/05/99) 0.060 0.054 0.384 0.091 9 BOI CH1/50 (27/11/00) 0.060 0.055 0.236 0.092 10 BOU NA 1/50 (23/12/99) 0.0700.067 0.119 0.081 11 CAM GE 1/50 (27/11/00) 0.063 0.072 0.079 0.077 12COU DI 1/50 (16/09/98) 0.094 0.073 0.081 2.468 COU DI 1/50 (09/03/00)0.074 0.070 0.077 1.935 COU DI 1/50 (08/09/00) 0.058 0.059 0.059 2.64113 CUC JE 1/50 (28/09/00) 0.059 0.056 0.173 0.088 14 DAN NO 1/50(28/09/00) 0.073 0.066 0.083 0.094 15 DAR DO 1/50 (10/03/94) 0.078 0.0660.795 0.085 16 DOM JO 1/50 (13/07/92) 0.060 0.057 0.275 0.096 17 ESM GU1/50 (18/12/00) 0.061 0.064 0.083 0.119 18 ETC MA 1/50 (25/09/98) 0.1140.067 1.266 0.089 ETC MA 1/50 (13/06/00) 0.057 0.057 0.854 0.084 19 FERJE 1/50 (26/06/00) 0.064 0.070 0.079 0.204 20 FIN RO 1/50 (13/11/00)0.063 0.065 0.097 0.089 21 FON RE 1/50 (29/06/00) 0.083 0.053 0.0950.070 22 GEM SA 1/50 (20/10/98) 0.093 0.071 0.090 0.086 GEM SA 1/50(29/04/99) 0.060 0.061 0.114 0.080 GEM SA 1/50 (07/06/00) 0.078 0.0500.079 0.068 23 GOU JE 1/50 (13/06/00) 0.069 0.052 0.098 0.072 24 KAZ AL1/50 (20/01/01) 0.059 0.065 0.117 0.080 25 KOH MI 1/50 (05/08/94) 0.0560.056 0.869 0.088 26 MAN GU 1/50 (06/04/00) 0.074 0.053 0.072 0.079 27MART DO 1/50 (03/05/00) 0.082 0.064 0.311 0.077 MART DO 1/50 (09/05/94)0.082 0.067 0.329 0.079 28 MART PA 1/50 (13/06/00) 0.055 0.058 0.1060.083 29 MEC EV 1/50 (28/01/00) 0.068 0.054 0.086 0.074 30 MEN CH 1/50(09/03/00) 0.051 0.053 0.078 0.155 31 MOR AN 1/50 (20/04/00) 0.075 0.0650.109 0.086 32 PAT MA 1/50 (09/07/98) 0.086 0.072 0.116 0.085 PAT MA1/50 (07/07/00) 0.054 0.053 0.104 0.070 PAT MA 1/50 (11/06/91) 0.0820.067 0.152 0.067 33 REF EL 1/50 (28/09/00) 0.070 0.067 0.085 1.153 34RIG ST 1/50 (02/02/98) 0.089 0.070 0.131 0.096 35 RIO EM 1/50 (11/06/92)0.071 0.067 0.115 0.073 36 ROB IS 1/50 (26/06/00) 0.054 0.056 0.0950.068 37 ROD CH 1/50 (12/04/99) 0.057 0.055 0.155 0.068 38 SAN FR 1/50(19/06/00) 0.059 0.054 0.139 0.088 39 SAU CH 1/50 (20/12/91) 0.074 0.0690.092 0.640 SAU CH 1/50 (27/11/00) 0.059 0.054 0.142 0.307 40 SEN BE1/50 (22/02/91) 0.058 0.057 0.143 0.082 41 SIL RE 1/50 (25/08/00) 0.0560.057 0.131 0.107 42 SIMO FR 1/50 (29/06/00) 0.061 0.055 0.221 0.077 43SLI ZB 1/50 (13/11/00) 0.065 0.055 0.260 0.095 44 SOL EM 1/50 (27/11/00)0.056 0.059 0.180 0.143 45 TAR AL 1/50 (24/07/91) 0.059 0.058 0.2030.414 46 TEM ST 1/50 (10/05/93) 0.059 0.055 0.219 0.338 TEM ST 1/50(18/11/99) 0.056 0.060 0.104 0.103 47 TER YV 1/50 (02/06/99) 0.061 0.0570.123 0.101 TER YV 1/50 (28/10/99) 0.104 0.076 0.122 0.412 48 THI SY1/50 (05/06/00) 0.056 0.062 0.096 0.089 49 THO EL 1/50 (04/12/00) 0.0770.066 0.115 1.189 50 THO MI 1/50 (08/06/00) 0.057 0.057 0.146 0.078 51THO PA 1/50 (04/12/00) 0.075 0.073 0.088 0.080 52 TOC RO 1/50 (23/11/92)0.058 0.056 0.095 0.097 53 TOC UR 1/50(23/11/92) 0.064 0.057 0.106 0.13654 VAL MAR 1/50 (12/02/92) 0.060 0.058 0.098 0.084 55 VIA JE 1/50(01/07/93) 0.098 0.077 1.426 0.099 VIA JE 1/50 (23/03/00) 0.059 0.0530.181 0.073

Thus, from reading the above table it seems that none of the sera testedhas any anti-R7V antibodies of type IgG₁ or IgG₂. Of 28 differentR7V-positive patients, 13 patients are IgG₃-positive (46.4%), 3 patientsare IgG₃-positive and IgG₄-positive (10.7%) and 12 patients areIgG₄-positive (42.8%).

In any one patient, the same anti-R7V IgG subclass is always found,regardless of the date on which the sample is taken, when the responseis positive (BER YV, COU DI, ETC MA, MART DO, SAU CH).

The subclass of antibodies directed against the envelope glycoprotein ofthe virus (gp160) was determined by means of an ELISA test (Table 3).TABLE 3 Determination of the anti-gp160 IgG subclass (Sanofi DiagnosticsPasteur Kit) Positivity threshold = 0.09 IgG₁ IgG₂ IgG₃ IgG₄ Blank 0.0650.062 0.060 0.067 Seronegative control 1/5 0.058 0.059 0.066 0.069 ARGCH 1/5 (30/11/00) 0.355 0.061 0.328 0.076 AUR PH 1/5 (03/01/94) 1.6450.068 0.160 0.781 BER YV 1/5 (25/11/99) 1.322 0.073 0.371 0.078 CAM GE1/5 (27/11/00) 0.121 0.050 0.066 0.051 COU DI 1/5 (16/09/98) 1.659 0.0640.479 0.162 DAN NO 1/5 (28/09/00) 0.855 0.058 0.361 0.069 DAR DO 1/5(31/08/98) 1.102 0.064 0.107 0.101 ESM GU 1/5 (18/12/00) 0.435 0.0650.084 0.111 ETC MA 1/5 (13/06/00) 0.513 0.060 0.135 0.172 FIN RO 1/5(13/11/00) 0.735 0.063 1.538 0.082 GEM SA 1/5 (07/06/00) 0.540 0.0580.104 0.081 MEN CH 1/5 (09/03/00) 1.260 0.054 0.209 0.069 PAT MA 1/5(09/07/98) 0.828 0.061 0.168 0.125 REF EL 1/5 (28/09/00) 1.511 0.0730.252 0.080 ROB IS 1/5 (26/06/00) 0.182 0.049 0.076 0.052 ROD CH 1/5(12/04/99) 0.952 0.059 0.154 0.071 SAN FR 1/5 (19/06/00) 1.112 0.0630.157 0.076 SAU CH 1/5 (27/11/00) 0.122 0.06 0.062 1.297 SOL EM 1/5(27/11/00) 0.191 0.056 0.106 0.062 TEM ST 1/5 (18/11/99) 0.834 0.0610.069 0.082 TER YV 1/5 (28/10/99) 1.389 0.061 0.252 0.231 THI SY 1/5(05/06/00) 0.633 0.063 0.088 0.088 THO EL 1/5 (04/12/00) 0.899 0.0570.158 0.064 THO MI 1/5 (08/06/00) 0.151 0.053 0.082 0.059 THO PA 1/5(04/12/00) 0.435 0.066 0.071 0.097 TOC RO 1/5 (25/11/99) 1.042 0.060.173 2.387 VIA JE 1/5 (01/07/93) 1.748 0.080 3.062 0.127

From reading this table, it seems that none of the sera tested has anyanti-gp160 antibodies of type IgG₂. Of 27 different patients, 27patients are IgG₁-positive (100%), 6 patients are only IgG₁-positive(22.2%), 10 patients are IgG₁-positive and IgG₃-positive (40.7%), 2patients are IgG₁-positive and IgG₄-positive (7.4%) and 9 patients areIgG₁-positive, IgG₃-positive and IgG₄-positive (29.6%).

This experiment confirms the dispersion of the response in terms ofantibodies directed against the envelope glycoprotein gp160 of thevirion, and the constancy of the IgG₃s or IgG₄s in protectedindividuals.

Example 2 Immunoprecipitation of HIV with Total IgG₃s, Purified fromNon-Progressor HIV-Positive Patients

2.1. Experimental Protocol:

A desired amount of pure HIV virus is added to a given amount of IgG₃and the mixture is left to incubate for 1 hour at +4° C.

The following are then added:

-   -   either 50-100 μL of protein G bound to magnetic beads, and        incubation is carried out for 1 hour at +4° C. (protein G binds        the IgG₁, IgG₂, IgG₃ and IgG₄ antibodies)    -   or 50-100 μL of protein A bound to magnetic beads, and        incubation is carried out for 1 hour at +4° C. (protein A binds        the IgG₁, IgG₂ and IgG₄ antibodies but not the IgG₃s).

The beads fix to that wall of the tube which is closest to the appliedmagnetic field, and the protein G bead-IgG₃-virus complex can then beisolated. The virus bound to the antibodies is then demonstrated bymeasuring the viral protein P24 released after lysis of the virus fromthe immunoprecipitated virus (COULTER kit (6604535)) and/or by theRT-PCR technique for detecting viral RNA. (See FIG. 2).

2.2. Results

A typical experiment is shown in Table 4 where 65% of the virus isprecipitated with 20 μg of IgG₃ from a non-progressor HIV-positivepatient, whereas the IgG₃s of an HIV-negative individual have no effect.TABLE 4 Immunoprecipitation (virus NDK/PBL of 29/02/96) % precipi-Sample tation 20 μg ARG CH IgG3 + 50 μL pure virus + 100 μL protein G64.70% 10 μg ARG CH IgG3 + 50 μL pure virus + 50 μL protein G 56.40% 20μg Seroneg. IgG3 + 50 μL pure virus + 100 μL protein G 0.40% 10 μgSeroneg. IgG3 + 50 μL pure virus + 50 μL protein G 0.20% virus control:50 μL pure virus + 50 μL protein G 0.10% 20 μg ARG CH IgG3 + 50 μL purevirus + 100 μL protein A 0.70% 10 μg ARG CH IgG3 + 50 μL pure virus + 50μL protein A 0.70% 20 μg Seroneg. IgG3 + 50 μL pure virus + 100 μLprotein A 0.20% 10 μg Seroneg. IgG3 + 50 μL pure virus + 50 μL protein A0.40% virus control: 50 μL pure virus + 50 μL protein A 0.40%

FIG. 3 shows the immunoprecipitation of the virus with the IgG₃s ofthree different patients. This immunoprecipitation of the virus for afixed amount of virus is dose-dependent. FIG. 4 shows that with a fixedamount of IgG₃, it is possible to trap various virus dilutions. FIG. 5shows that, regardless of the type of HIV, it is immunoprecipitated withthe IgG₃s, here from three patients. The viral strains used are primaryisolates having as phenotype clades A to F or a strain YBF 30 which isneither O nor M.

These experiments confirm that it is possible to isolate a virus byimmunoprecipitation from IgG₃s purified by protein G.

Example 3 Neutralization of HIV by Total IgG₃s, Purified fromNon-Progressor HIV-Positive Patients

3.1. Experimental Protocol:

50 μL of IgG₃ solution+50 μL of diluted virus (in 10% RPMI medium) areincubated for 1 hour at 37° C. in a 96-well plate. Then, 0.3×10⁶ cellsper well are added to the mixture and placed at 37° C. for 1 hour.Following two washes with 0% RPMI medium, the cells are placed in a24-well plate in 10% RPMI medium in the presence of IgG₃s (FIG. 6).

3.2. Results (cf. Table 5) TABLE 5 Neutralization of NDK/PBL (29/02/96)by IgG₃s or IgG_(1,2,4)s of ARG CH, ETC MA, AUR PH and THO MI. Dose(μg/mL) Day 3 Day 4 Day 5 Day 6 ARG CH IgG₃   40 μg/mL − (+) ++ ++T −(+) ++ ++T   20 μg/mL − − (+) + − − + ++T   10 μg/mL (+) + ++ ++T (+) +++ ++T   5 μg/mL (+) − (+) + (+) (+) ++ ++T ARG CH IgG_(1,2,4)   40μg/mL − (+) + ++T − (+) ++ ++T   20 μg/mL (+) + ++ ++T − (+) ++ ++T   10μg/mL + ++ ++ ++T − (+) + ++T   5 μg/mL (+) + ++ ++T (+) + ++ ++T ETC MAIgG₃  100 μg/mL (+) + ++ ++T (13/09/01) − (+) ++ ++T   50 μg/mL (+) + ++++T − (+) ++ ++T   25 μg/mL (+) ++ + ++T (+) + ++ ++T 12.5 μg/mL − − ++++T (+) (+) ++ ++T ETC MA IgG_(1,2,4)  100 μg/mL (+) ++ ++ ++T(13/09/01) (+) + ++ ++T   50 μg/mL (+) ++ ++ ++T (+) + + ++T   25 μg/mL(+) (+) ++ ++T − − ++ ++T 12.5 μg/mL (+) ++ ++T ++T − (+) ++ ++TPositive control 1/50 − − − − serum for the − − − − neutralization 1/100− − − − − − − − CONTROL MT4 − − − − cells − − − − VIRUS NDK/PBL 10⁻⁴(+) + ++ ++T (29/02/96) (+) + ++ ++T−: absence of syncitia+: presence of syncitia

The IgG₃S isolated from two patients neutralize HIV as shown in Table 5.With 40 μg and 20 μg of IgG₃, a neutralization of the virus is observed,that is to say an absence of syncitia, whereas the IgG_(1,2,4)s arelimited to the dose of 40 μg/mL.

Example 4 Virolysis of HIV by the Complement and Total IgG₃s, Purifiedfrom Non-Progressor HIV-Positive Patients

4.1. Experimental Protocol:

10 or 20 μg of IgG3+12.5 μL of pure virus are incubated for 1 hour at 4°C. with rotary stirring. Then, 1 mL of rabbit complement at {fraction(1/12)} (dilution of the complement in 10% RPMI medium heated to 37° C.)is added to the mixture, and the whole is incubated for 30 minutes at37° C. The samples are diluted and P24 is determined (COULTER kit6604535).

4.2. Results: (Table 6) TABLE 6 Test for lysis by the complement (virusNDK/PBL of 29/02/96) Sample % lysis   20 μg ARG CH IgG3 + 12.5 μLvirus + 1 mL 57.60% complement at {fraction (1/12)}   10 μg ARG CHIgG3 + 12.5 μL virus + 1 mL 60.30% complement at {fraction (1/12)} 12.5μL virus + 1 mL 10% RPMI, no triton 18.60% 12.5 μL virus + 1 mLcomplement at {fraction (1/12)}  1.70% 12.5 μL virus + 1 mL 10% RPMI +triton   100%

The complement binds to the IgG₃-virus complex and lyses the virus, asshown in Table 6 where 60% of the virus is lysed in the presence of 10μg of IgG₃.

The neutralization of viruses and virolysis in the presence ofcomplement give IgG₃s great potential for use as therapeutic antibodiesin HIV infection or in any other viral infection in which IgG₃s will bepresent either in the conventional manner or in the form of the variant.

This study suggests that the complement in addition with the anti-R7Vantibodies in the blood of people infected with HIV can lyse the virusand destroy its infectiousness.

The presence of infectious viruses in the plasma is 10 to 100 timesgreater in progressors than in non-progressors. This is because, sincethe progressor patients have no anti-R7V antibodies and hence noanti-R7V IgG₃s, there will be no neutralization or virolysis and thuspresence of viruses.

In conclusion, the inventors have shown that the antibodies fortherapeutic use of IgG₃ type which bind the complement and ensurevirolysis will be very effective not only in precipitating andneutralizing the virus but also in destroying it.

BIBLIOGRAPHIC REFERENCES

-   Coligan et al., Current Protocols in Immunology [updated annually]    (4 volumes), published by the “National Institutes of Health” by    Wiley Interscience.-   Harlow et al., (1988) Antibodies: A Laboratory Manual, Cold Spring    Harbor Publications, pp. 726.-   Köhler and Milstein (1975) Nature, 256: 495-497.-   Sambrook, J., Frischt, E. F. and Maniatis, T., Molecular cloning. A    Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring    Harbor, N.Y., 1989.

1-25. (canceled)
 26. A purified and/or isolated human immunoglobulin ofclass IgG3, or a fragment thereof, having the following characteristics:a life span in the serum of a patient of at least one month; and a heavychain, the molecular weight of which, determined by electrophoreticmobility, is less than the molecular weight of the IgG3s normallypresent in human serum (60 kDa), said heavy chain having a molecularweight of about 50 kDa and comprising the complement binding site ofsaid immunoglobulin.
 27. An Immunoglobulin according to claim 26, havinga concentration in the serum which is at least three times as great(i.e., 1 g/L) as the IgG3 concentration of a normal serum.
 28. AnImmunoglobulin according to claim 26, isolated and/or purified from apatient suffering from a pathology selected from the group consisting ofvirus infections, infectious diseases of variable development such asneurological diseases, skin diseases, auto-immune diseases and cancersof retroviral origin.
 29. An Immunoglobulin according to claim 28,wherein said virus is selected from the group consisting of humanimmunodeficiency virus (HIV), human T-cell leukaemia viruses (HTLVs),the cytomegalovirus (CMV), herpes viruses (HSV-1, HSV-2), theEpstein-Barr virus (EBV) and hepatitis viruses (HBV, HBC).
 30. AnImmunoglobulin according to claim 29, wherein said virus is HIV and saidpatient is a non-progressor seropositive patient.
 31. An Immunoglobulinaccording to claim 30, which selectively binds the R7V epitope of thegp160 protein of the HIV-1 virus, said epitope comprising the sequenceArg-Thr-Pro-Lys-Ile-Gln-Val.
 32. An immunoglobulin according to claim26, wherein said immunoglobulin or a fragment thereof is directly orindirectly labelled by a marker selected from radioactive isotopes,enzymes, dyes, haptenes, luminescent agents, and ligands.
 33. Animmunoglobulin according to claim 32, wherein said luminescent agent isselected from the group consisting of radioluminescent,chemiluminescent, bioluminescent, fluorescent and phosphorescent agents.34. An immunoglobulin according to claim 32, wherein said ligand isselected from the group consisting of biotin, avidin, streptavidin, anddigoxigenin.
 35. An immunoglobulin according to claim 32, which it iscoupled to a solid support either directly or indirectly via a spacerarm.
 36. An immunoglobulin according to claim 26, which is conjugateddirectly or indirectly to at least one agent selected from the groupconsisting of an antiproliferative agent, an antineoplastic agent and acytotoxic agent.
 37. An antiserum comprising an immunoglobulin accordingto claim
 26. 38. A method for the diagnosis of a pathology selected fromviral infections and infectious diseases of variable development,comprising the steps of: (i) contacting a sample of body fluid and/orbody tissue from a patient in which the presence of said pathology issuspected, with an immunoglobulin of class IgG3, or a fragment thereof,having the following characteristics: a life span in the serum of thepatient of at least one month; and a heavy chain, the molecular weightof which, determined by electrophoretic mobility, is less than themolecular weight of the IgG3s normally present in human serum (60 kDa),said heavy chain having a molecular weight of about 50 kDa andcomprising the complement binding site, or one of the fragments thereof,or an antiserum comprising the immunoglobulin; and (ii) detecting thepresence of an immune complex between said immunoglobulin and a specificepitope of said pathology.
 39. A method for the diagnosis of the humanacquired immune deficiency syndrome (AIDS), comprising the steps of: (i)contacting a sample of body fluid and/or body tissue, from a patient inwhich the presence of HIV is suspected, with an immunoglobulin of classIgG3, or a fragment thereof, having the following characteristics: alife span in the serum of the patient of at least one month; and a heavychain, the molecular weight of which, determined by electrophoreticmobility, is less than the molecular weight of the IgG3s normallypresent in human serum (60 kDa), said heavy chain having a molecularweight of about 50 kDa and comprising the complement binding site, orone of the fragments thereof, or an antiserum comprising theimmunoglobulin, or with an antiserum comprising said immunoglobulin; and(ii) detecting the presence of an immune complex between saidimmunoglobulin and the R7V epitope of the gp160 protein of the HIVcontained in said sample.
 40. A method according to claim 39, whereinthe detecting is carried out by an immunological test selected from thegroup consisting of an ELISA test, RIA, a sandwich method, animmunoprecipitation and an agglutination test.
 41. An in vitro methodfor treating biological fluid of a patient suffering from a pathologyselected from viral infections and infectious diseases of variabledevelopment, said method comprising the step of contacting saidbiological fluid with an effective amount of an immunoglobulin of classIgG3, or a fragment thereof, having the following characteristics: alife span in the serum of the patient of at least one month; a heavychain, the molecular weight of which, determined by electrophoreticmobility, is less than the molecular weight of the IgG3s normallypresent in human serum (60 kDa), said heavy chain having a molecularweight of about 50 kDa and comprising the complement binding site, or anantiserum comprising the immunoglobulin, so as to neutralize a specificepitope of the pathology in said fluid.
 42. An in vitro method accordingto claim 41, wherein the patient is suffering from AIDS.
 43. An in vitromethod for neutralizing cells infected with HIV in a biological sampleof body fluid and/or body tissue from a seropositive patient, comprisingthe following steps: a) combining, in a container, simultaneously,separately or sequentially, to form a reaction mixture: theimmunoglobulin according to claim 30, or an antiserum containing theimmunoglobulin, the biological sample which contains cells infected withHIV having the gp160 protein at their surface, and protein G bound tomagnetic particles; b) incubating the reaction mixture obtained in a)under conditions which allow the binding of said immunoglobulin to thegp160 protein, and preferably to the R7V epitope, so as to form acomplex, said complex comprising said immunoglobulin bound to anHIV-infected cell fixed to the magnetic particle; and c) displacing themagnetic particle to a predetermined point of the container containingthe reaction mixture, such that the displacement is brought about by amagnetic field acting on said magnetic particle.
 44. An in vitro methodaccording to claim 43, wherein said immunoglobulin binds the R7V epitopeof the gp160 protein.
 45. An in vitro method for diagnosing anon-progressor patient suffering from a pathology selected from viralinfections and infectious diseases of variable development, comprisingobtaining a biological tissue or fluid sample from the patient anddetecting the presence of immunoglobulin IgG3, said immunoglobulin IgG3having the following characteristics: a life span in the serum of thepatient of at least one month; and a heavy chain, the molecular weightof which, determined by electrophoretic mobility, is less than themolecular weight of the IgG3s normally present in human serum (60 kDa),said heavy chain having a molecular weight of about 50 kDa andcomprising the complement binding site.
 46. A method according to claim45, wherein said viral infection is AIDS.
 47. A pharmaceuticalcomposition for the treatment or prevention of a pathology selected fromviral infections and infectious diseases of variable development,comprising an immunoglobulin according to claim 26, or an antiserumcontaining said immunoglobulin, and a pharmaceutically acceptablecarrier, diluent or excipient.
 48. A method for treatment or preventionof a pathology selected from viral infections and infectious diseases ofvariable development, comprising administering the composition of claim47 to a patient suffering from the infection or disease.
 49. A methodaccording to claim 48, wherein the patient is suffering from AIDS.
 50. Amethod according to claim 49, wherein said immunoglobulin neutralizesHIV gp160 protein.
 51. A method according to claim 47, wherein saidpatient is a progressor patient.
 52. A composition comprising animmunoglobulin or fragment thereof according to claim 26, or anantiserum containing said immunoglobulin, and at least otheranti-retroviral agent selected from the group consisting of reversetranscriptase inhibitors and viral antiprotease inhibitors.